PROJECT SUMMARY/ABSTRACT This research program is focused on developing new strategies for the discovery of metalloenzyme inhibitors. Metalloenzymes are essential to numerous biological processes and are relevant to treating diseases, including cancer, bacterial/viral infections, hypertension, and others. Despite the prevalence of metalloenzymes (>40% of all enzymes are metalloenzymes) and their critical role in disease proliferation, the development of new metalloenzyme inhibitors is extremely underexplored. The PI (Cohen) has developed a research program that combines the principles of bioinorganic with medicinal chemistry and is widely recognized as one of the few efforts focused on the challenges of metalloenzyme inhibition. Small molecules that inhibit metalloenzymes utilize a metal-binding pharmacophore (MBP) functional group to bind to the active site metal ion(s) in the target. In the last project period, a focused MBP fragment library for use in fragment-based drug discovery (FBDD) against metalloenzymes was assembled. In this renewal application, the drug-like features of these MBP fragments will be improved by the application of isostere replacement. This is expected to yield new chemical matter for identifying metalloenzyme inhibitors, while accessing a wider range of physicochemical properties (e.g., acidity, lipophilicity) in these scaffolds. These metal-binding isosteres (MBIs) will then be used to improve a class of highly active inhibitors developed during the last project period against the influenza N-terminal endonuclease domain of the polymerase acidic protein (PAN). Although active against PAN endonuclease, the poor uptake properties of these inhibitors have led to suboptimal activity against the virus in cells. MBIs will be used to improve physicochemical properties, while retaining enzyme-based activity, to produce highly active inhibitors against the virus in live cells. Finally, to examine the on-target activity and selectivity of metalloenzyme inhibitors, our MBPs, MBIs, and PAN inhibitors will be examined by Cellular Thermal Shift Assay (CETSA) and affinity chromatography. These experiments will verify target engagement and evaluate how selectivity improves as the MBP is developed into a full-length PAN endonuclease inhibitor. Detailed cellular target engagement data using these methods for metalloenzyme inhibitors is scarce; therefore, these studies will be valuable for clarifying the selectivity, and hence the clinical prospects, of these therapeutic compounds. The previous project period generated many collaborations, patent disclosures, conference proceedings, and ~13 publications. In addition, skilled trainees for the biotechnology workforce were mentored, and translation of our results into startup companies was achieved. We will continue to nurture collaborations to discover best- and first-in-class metalloenzyme inhibitors that have the potential to improve human health. Overall, this research program will continue to play a leading role in identifying small molecule inhibitors for a challenging target class that has great merit for improving human health.